Direct interaction of proliferating cell nuclear antigen with the small subunit of DNA polymerase δ
Direct interaction of proliferating cell nuclear antigen with the small subunit of DNA polymerase δ
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DOI：
10.1074/jbc.m200065200
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发表时间：
2002-07-05
影响因子：
4.8
通讯作者：
So, AG
中科院分区：
文献类型：
--
作者：
Lu, XQ;Tan, CK;So, AG
增殖细胞核抗原（PCNA）和DNA聚合酶5之间的相互作用对于DNA复制/修复期间的进行性DNA合成是必不可少的;然而，直接与PCNA相互作用的DNA聚合酶5的亚基的身份至今尚未解决。在本研究中，我们已经使用了互惠共免疫沉淀实验，以确定核心DNA聚合酶5，125-kDa的催化亚基或50-kDa的小亚基，直接与PCNA相互作用的两个亚基。我们发现，PCNA共免疫沉淀与人类p50，以及小牛胸腺DNA聚合酶5异源二聚体，但不单独与p125，表明PCNA直接与p50，但不与p125相互作用。在p50的N末端发现了一个与噬菌体RB 69 DNA聚合酶的滑动夹结合基序相似的PCNA结合基序。一个22个氨基酸的寡肽含有这个序列（MRPFL）显示结合PCNA的远Western分析和竞争与p50结合PCNA的免疫共沉淀实验。p21抑制p50与PCNA的结合，表明两种蛋白竞争PCNA上的同一结合位点。这些结果表明，PCNA与DNA聚合酶5的相互作用是通过该酶的小亚基介导的。
The interaction between proliferating cell nuclear antigen (PCNA) and DNA polymerase 5 is essential for processive DNA synthesis during DNA replication/repair; however, the identity of the subunit of DNA polymerase 5 that directly interacts with PCNA has not been resolved until now. In the present study we have used reciprocal co-immunoprecipitation experiments to determine which of the two subunits of core DNA polymerase 5, the 125-kDa catalytic subunit or the 50-kDa small subunit, directly interacts with PCNA. We found that PCNA co-immunoprecipitated with human p50, as well as calf thymus DNA polymerase 5 heterodimer, but not with p125 alone, suggesting that PCNA directly interacts with p50 but not with p125. A PCNA-binding motif, similar to the sliding clamp-binding motif of bacteriophage RB69 DNA polymerase, was identified in the N terminus of p50. A 22-amino acid oligopeptide containing this sequence (MRPFL) was shown to bind PCNA by far Western analysis and to compete with p50 for binding to PCNA in co-immunoprecipitation experiments. The binding of p50 to PCNA was inhibited by p21, suggesting that the two proteins compete for the same binding site on PCNA. These results establish that the interaction of PCNA with DNA polymerase 5 is mediated through the small subunit of the enzyme.